Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin ␣-subunit were mostly unaffected. Outer segment membranes of GC1 ؊/؊ and GC double knock-out cones were destabilized and devoid of cone transducin (␣-and ␥-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the downregulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.

Mouse photoreceptor function and survival critically depend on Ca2+-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12 and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5 to 28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 μM cGMP s−1 or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca2+ in the presence of GCAP1 (EC50Ca ~132-139 nM) than GCAP2 (EC50Ca ~50-59 nM), thus arguing that Ca2+ sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T␣-subunit, cone phosphodiesterase 6␣Ј (PDE6␣Ј), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T␣ and cone PDE6␣Ј were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65 Ϫ/Ϫ Rho Ϫ/Ϫ double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.

We discuss putative mechanisms of membrane protein transport in photoreceptors based on Pde6d and Gucy2e/Gucy2f knockout mice. Knockout of the Pde6d gene encoding PrBP/delta, a prenyl binding protein present in the retina at relatively high levels, was shown to impair transport of G-protein coupled receptor kinase 1 (GRK1) and cone phosphodiesterase alpha' subunit (PDE6alpha') to the rod and cone outer segments. Other prenylated proteins are minimally affected, suggesting some specificity of interaction. Knockout of the Gucy2e gene encoding guanylate cyclase 1 (GC1) disrupted transport of G-protein coupled receptor kinase 1 (GRK1), cone PDE6alpha', cone transducin alpha and gamma subunits (cTalpha and cTgamma) to the cone outer segments, while a GC1/GC2 double knockout prevented transport of rod PDE6, but not transducin, GRK1, or rhodopsin, to the rod outer segments. These knockout phenotypes suggest that PrBP/delta functions in extracting prenylated proteins from the endoplasmic reticulum (ER) where they dock after prenylation, and that GC-bearing membranes may co-transport peripheral membrane proteins in vesicles. We conclude that distinct pathways have evolved in rods and cones for transport of integral and peripherally membrane-associated proteins.